Introduction

Alzheimer’s disease (AD) is a progressive neurological disorder marked by cognitive decline, behavioral abnormalities, memory loss, and intellectual deterioration1,2. Despite significant efforts, pharmaceutical therapies for AD have not achieved widespread success, mainly due to their focus on the amyloid hypothesis. This hypothesis suggests that the accumulation of amyloid β (Aβ) and phosphorylated tau proteins triggers a cascade of neurotoxic events, including oxidative stress, inflammation, and neurotransmitter imbalance, ultimately leading to synaptic dysfunction and impaired synaptic plasticity, particularly in the hippocampus3,4,5,6,7.

Photobiomodulation therapy (PBMT) is a non-pharmacological, non-invasive method using light sources like lasers and LEDs for therapeutic purposes, including enhancing neuronal synaptic function8,9,10. Studies have shown that low-level LED therapy, particularly at 40 Hz, can reduce AD neuropathology11,12,13,14,15,16,17. This frequency of light flickering has been demonstrated to reverse AD pathological features in animal models11,14,17, leading to the 2021 US FDA approval for further clinical investigation18. In animal studies, 40 Hz flickering LED light affects neuronal activity through various systems and pathways, primarily the visual system and possibly the skin. When exposed to 40 Hz light, photoreceptors in the retina are activated, sending signals to the brain that promote gamma oscillations in the visual cortex and the hippocampus. While some evidence suggests that skin photoreceptors might also respond to light and influence sensory processing and behavior, most research highlights the importance of visual pathways. At the cellular level, 40 Hz light exposure has been shown to decrease inflammation, maintain brain myelination, protect oligodendrocytes, and enhance synaptic health19,20,21,22.

The accumulation of Aβ peptides in AD results in mitochondrial dysfunction, abnormal Ca+2influx, synaptic loss, inflammation, and cell death23,24,25,26,27. PBMT has been shown to improve mitochondrial function by increasing ATP content and respiratory chain activity, particularly cytochrome c oxidase (complex IV)28,29. Our previous work demonstrated that 40 Hz flickering white LEDs restored mitochondrial function in a rat AD model by enhancing complexes I and IV activities and mitochondrial membrane potential16,30. Given the crucial role of mitochondria in synaptic transmission, ion/metabolite homeostasis, and cell survival, flickering 40 Hz light may hold therapeutic potential for AD by improving mitochondrial function31,32,33,34.

Mitochondrial ion channels, particularly mitochondrial large conductance voltage- and Ca2+-activated K+(mitoBKCa) channels, play a significant role in neuroprotection35,36,37. These channels help prevent mitochondrial Ca+2overload and reduce reactive oxygen species (ROS)36. In AD and diabetic models, Aβ(1–42) peptides impair ATP-sensitive mitoBKCa channels, reducing their conductance and open probability16,38,39,40. Independent studies suggest that 40 Hz LED light can enhance the activity of these channels in rat brains16. However, the expression and functionality of mitoBKCa channels in STZ toxicity models of AD and the effects of 40 Hz LED light on these channels require further investigation.

This study evaluated mitochondrial metabolomics in a rat model of STZ-induced AD. We also examined the effects of 40 Hz flickering LED light on the gating and permeability of mitochondrial mitoBKCa channels and their potential to prevent STZ-induced mitochondrial changes.

Materials and methods

Chemicals

Sucrose, D-mannitol, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), BSA (bovine serum albumin), EGTA (ethylene glycol-bis (β-aminoethyl ether)-N, N, N′, N′- tetraacetic acid), digitonin, nagarse protease, sodium bicarbonate, Trizma-HCl, potassium chloride, glibenclamide, ATP, Tris–HCl, EDTA, NADH, KCN (potassium cyanide), sodium dithionite, monopotassium phosphate (KH2PO4), ubiquinone-1 (coenzyme Q1), reduced cytochrome c, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), and rhodamine 123 (Rh 123) were obtained from Sigma (Sigma-Aldrich, St. Louis, MO, USA); n-decane was purchased from Merck (Merck KGaA, Dermstadt, Germany).

Animals

Seventy-two adult male Wistar rats (two months old) weighing 200–220 g were obtained from Tehran University, Tehran, Iran. Four rats were kept per cage under standard conditions, with a temperature of 23 ± 1 °C, humidity ranging from 50 to 60%, a 12-hour light/dark cycle, and free access to food and water. This study was approved by the ethics committee of the Tehran University of Medical Sciences (IR.TUMS.MSP.REC.1399.066). All experiments were conducted under the Guide for the Care and Use of Laboratory Animals, National Institute of Health.

Light flicker exposure apparatus

Over the course of 7 days, rats of specific experimental groups (see below) were exposed to flickering 40 Hz light (15 min/day) in a black box with 50 × 50 × 50 cm dimensions. The LED strip was positioned inside at the top of the box and emitted white light with a visible wavelength of 425–550 nm and an average irradiance/power density of 12 mW/cm2. White light was pulsed in a flickering 40 Hz pattern (12.5 ms on and 12.5 ms off). The LED on/off state was controlled through an AVR microcontroller circuit connected to a computer via a USB cable. Other characteristics of this approach included duty cycle (50%), average power (30 W), and average energy density (10.8 J/cm2). Animals were exposed to 40 Hz flickering light during the light phase of a 12-h light/dark cycle.

Experimental groups

Animals were randomly assigned to the following experimental groups: (1) sham: intracerebroventricular (ICV)-saline injection with no light treatment; (2) sham + light: ICV-saline injection with light treatment; (3) STZ (to induce AD phenotype): ICV-STZ injection with no light treatment; and (4) STZ + light: ICV-STZ injection with light treatment. Streptozotocin (STZ) was dissolved in distilled water and administered a single dose of 3 mg/kg. After anesthetizing the rats with a combination of ketamine (100 mg/kg) and xylazine (5 mg/kg), they were secured in a stereotaxic frame. A single bilateral intracerebroventricular (icv) injection of STZ (3 mg/kg, 2.5 µl per ventricle) was administered over 5 min on each side. The targeted coordinates were 0.8 mm posterior to the bregma, ± 1.5 mm lateral to the sagittal suture, and 3.6 mm below the dura mater. The STZ dose was selected based on previous studies using a rat model for sporadic Alzheimer’s disease41,42. Seven days following ICV-STZ/saline injections, rats of groups 2 and 4 underwent daily exposure to 40 Hz flickering white light for 15 min for 7 consecutive days. Tissue collection and electrophysiology experiments were performed at the end of the study. Details of the experimental design are outlined in Fig. 1.

Fig. 1
figure 1

Schematic diagram of the experimental design.

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Morris water maze

The Morris water maze (MWM) is one of the most widely used tools to assess spatial learning and memory in rodents43. Our design for the MWM task was a circular black tank (120 cm in diameter and 70 cm in height). It was placed in a dim room with visual cues. The tank was filled to a depth of 40 cm with water (22 ± 1 °C). The maze was divided into four equal quadrants with four starting lines: north, south, east, and west. A hidden circular platform (10 cm in diameter) was located in the quadrant west, submerged 1.5 cm beneath the water’s surface. The platform was invisible to rats. Also, the platform’s location did not change until the end of the test; fixed extra-maze visual cues were present at various locations around the maze. On the first day before initiation of the tests, in both light and dark environments, rats were placed on the platform for 30 s to get adjusted, and then the learning trials began. The task typically consists of two stages: the acquisition phase (assessment of spatial learning) and the probe test (evaluating memory retention and retrieval). MWM experiments were performed for four consecutive days. The acquisition phase comprised one session with four trials (30-second intervals) lasting 90 s for three consecutive days. Animals were subjected to a 60-second probe test (with a removed platform) 24 h after the last trial session. During each trial, the swimming paths of the animals were automatically recorded using a video camera-based system (EthoVision, Noldus, Version 14). The latency time to find the hidden platform (escape latency) was analyzed in the acquisition phase. Parameters assessed in the probe test included the latency of the first entry to the target quadrant, the number of times crossed to the platform, swimming speed (velocity), and time spent in the target quadrant. One rat in each group was identified as a ‘poor performer’ and excluded from the study.

Novel object recognition task

The Novel Object Recognition (NOR) test assesses the cognitive ability of experimental animals by measuring their natural tendency to explore a new object more than a familiar one44. The test was conducted over three days, beginning with a habituation period where the animals could explore an empty arena (60 × 60 × 50 cm) for 5 min. The animals were placed in the arena with two similar objects on the second day. Each rat was placed alone at the midpoint, next to the front wall of the box, opposite the objects, which they explored during a familiarization session. After 10 min, the rat was returned to its cage. On the third day, one of the familiar objects was replaced with a novel object, and the rat was allowed to explore the arena for a 10-minute break. Although the new object had the same height, material, and volume as the familiar object, it differed in shape and color. The animals ‘exploration of the objects was determined by the distance of their nose being within 2 cm of the object. It should be noted that the animals had not encountered these objects before they were examined. Active exploration of an object was defined as the rat’s nose being within 2 cm of the object, which was recorded and assessed using a video camera-based system (EthoVision, Noldus, Version 14). The Recognition Index (RI) was calculated based on the time spent investigating the novel object (TN) relative to that of the total object investigation (TF+TN).

Passive avoidance

A passive avoidance shuttle apparatus assessed memory retention deficits. The apparatus had light and dark compartments divided by a guillotine door, with a shock floor grid in the dark section39. When the rat entered the dark compartment, the door closed; after 30 s, it was returned to its cage. On the first day, three trials with 30-minute intervals were conducted: two for adaptation and one for training. The latency to enter the dark room was measured. During training, rats received a 1.5 s electric shock (50 Hz, 1 mA) upon entering the dark compartment. The next day, retention latency time and step-through latency (STL) were measured without a shock to evaluate memory retention (cutoff time: 600 s). Experiments occurred between 10.00 a.m. and 2.00 p.m.

Tissue collection for biochemical tests

After the behavioral tests were completed, rats were decapitated under ketamine (150 mg/ kg) anesthesia. Hippocampal tissue (n = 5 rats per group) was dissected and homogenized in cold RIPA buffer (pH 7.4). The supernatant was collected for biochemical tests after centrifugation (~ 10,000 g, 4 °C, 15 min). The Bradford assay determined the total protein concentration in each sample.

Serotonin and dopamine assays

According to the manufacturer’s instructions, dopamine and serotonin levels in hippocampal tissue homogenates were assessed using specific ELISA kits (Cat#: RK00642 and Cat#: TN2437, Zellbio, Germany).

Histological evaluation using Congo red stain

Animals (n= 6) were anesthetized using a high dose of ketamine (150 mg/kg) and transcardially perfused with heparinized normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4). The brains were removed, kept in 4% paraformaldehyde solution for 24 h, and prepared for Congo red staining by cutting the hippocampal block into 7 μm coronal Sects45,46,47. Plaques of the hippocampus area bilaterally were counted on every slide. The hippocampus’ cornu ammonis (CA1, CA2, and CA3) and dentate gyrus regions were subject to neuronal Aβ plaque counting. Each section was counted twice.

Isolation of mitochondria

The Navarro et al. protocol was utilized to isolate mitochondria48. Briefly, the whole brain from each animal was homogenized in a mitochondrial isolation buffer containing 230 mM mannitol, 1.0 mM EDTA, 70 mM sucrose, and 10 mM Tris–HCl at a pH of 7.40. The homogenized brains were centrifuged at 700 g for 10 min, after which the supernatant was separated and centrifuged at 8000 g for 10 min to obtain mitochondria pellets. The mitochondrial samples were stored at − 80 °C for further biochemical analysis. A Bradford assay determined Mitochondrial protein content (0.7 mg/ml)35.

Mitochondrial inner membrane isolation

To isolate the inner mitochondrial membrane, brain mitochondria were obtained 28 days post-ICV-STZ injection using a modified protocol49,50,51. Dissected brains were homogenized in a solution containing 225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, 1 mg/ml BSA at pH 7.4, and 0.05% nagarse. The homogenate was centrifuged at 2000 g for 4 min, and the supernatant was centrifuged at 12,000 g for 9 min to obtain a pellet. This pellet was dissolved in MSE-digitonin (0.02%) solution, then centrifuged at 12,000 g for 11 min. The final pellet was suspended in H2O (5 mg/ml), stirred for 20 min, homogenized (20x), and centrifuged twice at 12,000 g for 5 min. The resulting mitoplasts were treated with 0.1 M Na2CO3(pH 11.5) for 20 min, then centrifuged at 100,000 g for 30 min. The inner membrane vesicles were stored at − 80 °C until use51.

Extraction of L‑α‑phosphatidylcholine

L-α-phosphatidylcholine (L-α-lecithin) was utilized to create a planar lipid bilayer. The Singleton and Gray’s52 method was applied to extract L-lecithin from fresh egg yolk; the extracted lipid purity was verified using thin-layer chromatography (TLC), which revealed a purity of approximately 98% compared to standard lecithin.

Single-channel recordings

A Delrin partition with a 150-µm-diameter hole was used to create cis and trans chambers, representing cytoplasmic and luminal sides, respectively, to form planar phospholipid bilayers. Both chambers were filled with 4 ml of KCl (200 mM cis/50 mM trans), with pH adjusted to 7.2 with Tris-HEPES. A 25 mg/ml L-lecithin in n-decane solution was used to paint the bilayer, mechanically fused with a vesicle, by touching the membrane from the cis side with a stainless-steel wire. Ag/AgCl electrodes monitored the voltage, and a BC-525D amplifier (Warner Instrument, USA) in voltage-clamp mode amplified the current. The amplifier induced a low-frequency triangular wave (1–10 Hz, 5–20 mV) to estimate bilayer thickness, resulting in capacitance values of 200–300 pF. Single-channel recordings were filtered at 1 kHz, digitized at 10 kHz, and stored using Clampex10 (Axon Instruments Inc., USA), with analysis in pCLAMP10. Single-channel conductance was measured from the current-voltage curve slope, and channel open probability (Po) was calculated from 40-sec recording segments, presented as means ± S.E.

Field excitatory postsynaptic potential (fEPSP) and long-term potential

To record field excitatory post-synaptic potential (fEPSP), an extracellular bipolar stainless-steel electrode (0.125 mm diameter) was inserted into the medial perforant pathway (4.2 mm lateral to the lambda and − 3.2 mm ventrally), and another electrode was placed in a DG region with maximal response (-3.8 mm posterior and 2.2 mm lateral to the bregma). Extracellular field potentials were amplified 1000 times, digitized at 10 kHz, and filtered with a differential amplifier (0.1 Hz to 10 kHz). Biphasic square waves (200 ms width) were used as stimuli. Signals were transmitted to a computer via an A/D interface (Science Beam Co., Iran) and analyzed using eProbe software. LTP was induced by high-frequency stimulation (HFS) consisting of 10 sets of 10 pulses at 400 Hz, with a 10 s interval between sets. Baseline stimulation resumed post-HFS, and recordings continued for 60 min41.

Paired-pulse response

After establishing a baseline for 30 min, we measured the effect of paired-pulse stimulation on depression/facilitation. The stimulation was delivered at 20, 30, 50, 70, 100, and 120 ms intervals at 40% of the maximum stimulus intensity. Ten consecutive evoked responses were averaged for each paired-pulse stimulation. Various interstimulus intervals were used to determine the fEPSP slope ratio [percentage of the second fEPSP slope/first fEPSP slope; fEPSP2/fEPSP1%] and the population spike amplitude ratio [percentage of the second population spike amplitude relative to the first population spike amplitude; PS2/PS1%]41.

Tandem mass spectrometry (MS/MS) analysis

Using the Navarro et al. protocol48, isolated mitochondria were suspended in H2O (5 mg/ml) and stirred for 10 min, then homogenized 20 times using a glass homogenizer. Amino acid levels in the samples were assessed using isotope dilution tandem mass spectrometry (MS/MS, SHIMADZU, CLAM2040, JAPAN) and CHROMSYSTEM kits. Each 10 µl sample was mixed with 200 µl of internal standard, vortexed for 10 s, and centrifuged at 4 °C for 10 min at 10,000 rpm. The supernatant (150 µl) was dried for 20 min at 45 °C. After adding 50 µl of acetyl chloride 1-butanol and shaking for 1 min, the samples were incubated at 65 °C for 15 min, dried, and then mixed with 50 µl of 75% acetonitrile. Samples were shaken for 2 min and introduced to the MS/MS system with an injection volume of 10 µl, a flow rate of 150 µl/min, and a 2.074 min analysis time. Multiple reaction monitoring (MRM) was used as the scan mode53.

Complex I (NADH‑CoQ Oxidoreductase) activity assay

To determine the activity of complex I, a slightly modified version of the method described by Spinazzi et al.54 was used. Briefly, mitochondria were isolated and added to an assay medium of distilled water, 500 mM potassium phosphate buffer (pH 7.5), 10 mM KCN, 10 mM NADH, and 50 mg/ml BSA without fatty acids. The reaction was initiated by adding ubiquinone-1 (10 mM) as an electron acceptor, and the decrease in absorbance was measured at a wavelength of 340 nm for 5 min with 15-second intervals. NADH has an extinction coefficient of 6.2/mM/cm.

Complex IV (cytochrome c oxidase) activity assay

The activity of complex IV was determined using a previously described method54. In brief, the cytochrome oxidation process was initiated by adding brain mitochondrial preparations to the reaction buffer solution. The reduction in absorbance was measured at 550 nm with 15-second intervals for 5 min. The enzyme activity was expressed in nmol/min/mg of protein.

Mitochondrial membrane potential measurement

According to the protocol published by Luo and Shi’s55, the cationic fluorescent dye Rhodamine 123 (Rh123) was used to estimate mitochondrial membrane potential (MMP). In brief, mitochondrial fractions were incubated with Rh123 for 5 min and then excited at a wavelength of 490 nm. An emission wavelength of 530 nm was used to detect Rh123. MMP was assessed by analyzing the fluorescence intensity using a fluorescent microplate reader (Synergy™ HTX, Agilent Technologies, Inc. CA, USA).

Reactive oxygen species assay

DCFH-DA was utilized to measure ROS production, as it can easily cross membranes and transform into a highly fluorescent DCF when exposed to reactive oxygen during oxidation. The isolated mitochondrial samples (0.7 mg/ml protein) were incubated with DCFH-DA in microplate wells for 20 min at 25 °C. Fluorescence was measured using a Multi-Mode Microplate reader (Synergy™ HTX, Agilent Technologies, Inc. CA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm56.

Systems biology: metabolites-related pathway identification

To identify the biological pathways affected by the metabolites that were dysregulated by STZ (based on our results), we used enrichment, pathway, and joint pathway analyses through MetaboAnalyst 6 (https://www.metaboanalyst.ca/)57. Before performing the joint pathway analysis, we identified the proteins related to the metabolites (amino acids) of interest through KEGG58,59,60 and MetaCyc (databases integrated into the Pathview Web server (https://pathview.uncc.edu/))61.

Statistical analysis

Statistics were performed using Prism 8 software (Graph Pad Software, USA). All data analyses related to the experimental groups were carried out blindly. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s post hoc test to compare MWM in the acquisition phase, exploring time in the novel object test, and fEPSP results. In addition, one-way ANOVA followed by Bonferroni’s post hoc test was employed to compare more than two groups. The data are presented as mean ± S.E. Differences were considered statistically significant when P < 0.05.

Results

Cognitive function was improved by flickering 40 Hz white light in a rat model of AD

To assess cognition, memory, and learning and to evaluate the potential benefits of flickering 40 Hz white light, we conducted behavioral tests on the following experimental groups: sham, sham + light, STZ, and STZ + light.

Morris Water Maze (MWM) Test

The MWM test showed a significantly higher latency to reach the platform during the learning phase (days 1–3) in STZ-induced AD rats compared to sham groups (sham and sham + light; P < 0.0001 for both). Daily 15-minute exposure to flickering 40 Hz white light for 7 consecutive days significantly reduced the escape latency in STZ-induced AD rats (P < 0.01; Fig. 2A). All groups improved their performance from day 1 to day 3 of the training session. However, the performance gap between STZ (no intervention) and sham groups persisted throughout the experiment. By day 3, light exposure helped maintain the escape latency in STZ animals at near-control levels (STZ + light vs. sham + light).

Fig. 2
figure 2

Exposure to flickering 40 Hz white light improved cognitive function in a rat model of STZ-induced AD. MWM: Comparison of the time escape latency to reach the hidden platform in the acquisition phase over 3 days (A) and the time escape latency (B), total time spent in the target quadrant (C), and number of times crossing the platform (E) as assessed in the probe tests in sham, sham + light, STZ and STZ + light groups. Panel D shows a visual comparison of the average heatmap of MWM trials; the scale bar indicates the time spent in the target quadrant. Panels F and G depict the comparison of the exploring time of a novel object followed by a familiar object and the recognition index, respectively, in all 4 experimental groups. The effects of light treatment on passive avoidance learning acquisition in sham and STZ rats are indicated by differences in step through latency (STL; step-through latency) during the retrieval test performed 1 day after passive avoidance acquisition (H). One-way ANOVA analysis followed by Bonferroni’s post hoc test was performed to compare the mean between groups. Data are expressed as mean ± SEM (n = 7 rat/group). *P < 0.0.5, **P < 0.01, ***P < 0.001, **** P < 0.0001 compared to sham, and #P < 0.05, ##P < 0.01 compared to STZ.

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Probe trial test

Memory retrieval, assessed 24 h after the last training, showed a delayed first entry to the platform location in STZ-induced AD rats compared to sham groups (F(3, 24) = 42.73, P < 0.0001), partially improved by 40 Hz white light treatment (P < 0.001; Fig. 2B). Additionally, STZ rats spent less time in the target quadrant and crossed the platform fewer times than sham groups (Fig. 2C-E; P < 0.0001 for both). STZ + light rats significantly improved these cognition parameters compared to untreated STZ animals (P < 0.01 for both). No significant differences were observed between the sham and sham + light groups. No significant differences were observed between groups in swimming speed (data not shown).

Novel object recognition (NOR) Task

In the NOR task, STZ rats exhibited less exploration of the novel object compared to sham rats, as indicated by total exploration time (Fig. 2F; P < 0.001) and recognition index (Fig. 2G; P < 0.05). Flickering light treatment increased exploration time (P < 0.01 vs. STZ) and normalized the recognition index (P < 0.05 vs. STZ) to control levels. Light exposure did not affect the sham group’s total exploration time and recognition index (Fig. 2F and G).

Passive avoidance behavior

Passive avoidance behavior, measured by step-through latency (STL), was significantly reduced in STZ-treated rats compared to sham-treated rats (Fig. 2H; P < 0.0001). Flickering 40 Hz white light increased STL by approximately threefold in STZ animals (P < 0.01). STL was not affected by flickering light in the sham group (Fig. 2H). These results indicate that flickering 40 Hz white light improves recall abilities and cognitive functions in rats with STZ-induced AD.

Flickering 40 Hz white light prevented the deficits in serotonin levels and mitochondrial phenotype in STZ-induced AD rats

Serotonin and dopamine levels

Serotonin and dopamine levels were measured in hippocampal homogenates from sham, STZ, and STZ + light groups. Serotonin was significantly reduced in STZ rats compared to sham controls (P < 0.01), while dopamine levels showed no significant differences between groups. Treatment with flickering 40 Hz white light restored serotonin levels to control in STZ rats (P < 0.05; Fig. 3A).

Fig. 3
figure 3

Flickering 40 Hz white light prevented the deficits in serotonin levels and mitochondrial phenotype in the hippocampus of STZ-induced AD rats. Neurotransmitter (serotonin and dopamine) levels and mitochondrial phenotype were analyzed in hippocampal tissue homogenates obtained from sham, STZ, and STZ + light rats. Serotonin and dopamine levels were determined by ELISA assay (A; n = 6). Mitochondria were isolated for the measurement of complex I/IV activities, MMP, and ROS levels (B-E; n = 7). In addition, brain sections from sham, sham + light, STZ, and STZ + light rats were stained with Congo red, after which Aβ plaques were counted (F-G). One-way ANOVA analysis followed by the Bonferroni’s post hoc test was performed to compare the mean between groups. Data are expressed as mean ± SEM. *P < 0.0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to sham, and #P < 0.05, ## P < 0.01 compared to STZ.

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Mitochondrial phenotype

The mitochondrial phenotype was assessed by evaluating complex I and IV activities, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) levels in isolated mitochondrial fractions. Complex I and IV activities were significantly reduced in the hippocampus of STZ rats compared to the sham group (P < 0.0001). Flickering 40 Hz white light improved the activity of both complex I (P < 0.001) and IV (P < 0.05) in STZ rats (Fig. 3B, C).

Mitochondrial membrane potential and ROS production

STZ-induced AD rats exhibited a significant increase in MMP (ΔΨm) (P < 0.001), which was normalized by flickering 40 Hz white light treatment (Fig. 3D). Higher ROS production was observed in isolated hippocampal mitochondria of STZ rats compared to sham controls (P < 0.001). This ROS overproduction was absent in STZ rats with flickering 40 Hz white light (P < 0.01; Fig. 3E).

Flickering 40 Hz white light reduced Aβ plaque formation in STZ-induced AD rats

Aβ accumulation in the hippocampus’ cornu ammonis (CA1, CA2 and CA3) and dentate gyrus regions, a major pathological factor in AD progression, was assessed using Congo red staining. No differences in plaque abundance were observed between sham and sham + light animals. Significant Aβ plaque accumulation was evident in the hippocampus of STZ rats (P < 0.001 vs. sham), which was reduced by approximately 50% with flickering 40 Hz white light treatment (P < 0.01 vs. STZ; Fig. 3F and G).

Flickering 40 Hz white light prevented STZ-Induced deficits in electrophysiological properties of hippocampal neurons

To assess the long-term effects of 7-day treatment with flickering 40 Hz white light on neuronal activity, we evaluated basal synaptic transmission and plasticity in the perforant pathway of the hippocampus one month after STZ induction of AD. Basal synaptic transmission was analyzed using stimulus-response curves, and synaptic plasticity was assessed by paired-pulse facilitation (PPF) and long-term potentiation (LTP).

Basal synaptic transmission

The PS amplitude and slope of the excitatory postsynaptic potential were recorded in lateral perforant pathway-DG synapses of sham, STZ, and STZ + light animals. The input/output (I/O) curves revealed that PS amplitude increased with current intensity in all groups (100 to 1000 µA), (Fig. 4A).

Fig. 4
figure 4

Flickering 40 Hz white light prevented the STZ-induced deficits in electrophysiological properties of hippocampal neurons in rats. Basal synaptic transmission and plasticity in the perforant pathway of the hippocampus was assessed in sham, STZ, and STZ + light animals. (A) The acquired input/output (I/O) curve from a 60 s recording and single traces of the DG neurons’ reaction to numerous stimulus intensities (100 to 1000 µA)(n = 6). (B) The effect of flickering 40 Hz white light on LTP induction and maintenance in the DG up to 60 min after high-frequency stimulation (HFS). Data were normalized against the baseline period (-30 to + 60 min), and single traces were recorded before and after HFS. fEPSP slope change (%) and time following HFS. (C-D) The PS amplitude and slop mean were recorded at 0 to 60 min after applying HFS (n = 7). (E-F) In short intervals between two strictly paired stimuli, the amplitude of PS and fEPSP slope were prompted by a paired pulse in all groups. One-way or Two-way ANOVA analyses followed by the Bonferroni’s post hoc test were performed to compare the mean between groups. Data are expressed as mean ± SEM (n = 6 rat/group for B-E). *P < 0.0.5, ** P < 0.01, ***P < 0.001, ****P < 0.0001 compared to sham, and #P < 0.05, ##P < 0.01, ###P < 0.001 compared to STZ.

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Long-term potentiation (LTP)

After 60 min of LTP induction, STZ rats showed significantly lower fEPSP compared to sham controls (P < 0.01), which was normalized by flickering 40 Hz light treatment (Fig. 4B). High-frequency stimulation (400 Hz) of lateral perforant pathway-DG produced high PS amplitude and long-lasting synaptic potentiation, both suppressed in STZ rats (P < 0.0001 and P < 0.05, respectively). The light treatment restored these parameters to control levels (Fig. 4C and D).

Paired-pulse facilitation (PPF)

Short intervals between paired stimuli showed significantly lower PS amplitude in STZ rats compared to sham (P < 0.0006), improved by 40 Hz light treatment at 20ms intervals (Fig. 4E). Similarly, fEPSP slope was significantly lower in DG neurons of STZ rats (P = 0.049), but flickering light treatment prevented synaptic potentiation defects at 20, 30, and 50ms intervals (Fig. 4F).

Overall, the electrophysiological properties of hippocampal neurons, including synaptic transmission and plasticity, were disrupted in STZ-induced AD rats. These deficits were counteracted by flickering 40 Hz white light treatment initiated two weeks prior to assessments.

Induction of experimental AD reduced channel gating and conductance in mitochondrial mitoBKCa channels, but was prevented by 40 Hz light therapy

Next, we explored if the biophysical properties of mitochondrial mitoBKCa channels are affected by STZ injection and/or exposure to flickering light. Mitochondrial inner membranes were extracted (as vesicles) from the whole brain, and channel activities were analyzed. The success rate of mitoBKCa channel recordings was approximately 45%, 36%, and 40% for sham, STZ, and STZ + light rats, respectively.

Channel conductance in mitochondrial inner membrane vesicles from STZ rats showed a ~ 56% reduction (124 ± 7.2 pS) compared to sham controls (211 ± 6.4 pS, set as 100%; Fig. 5A C). In STZ + light animals, conductance increased to 194 ± 11.5 pS, normalizing to control levels. Channel gating behavior revealed voltage dependence, with longer silent periods at potentials below − 40 mV and outward currents above − 10 mV.

Channel open probability (Po) at + 40 mV was 0.9 ± 0.05 in sham rats, decreasing to 0.52 ± 0.04 in STZ rats. Flickering light treatment increased Po in STZ rats from 0.16 ± 0.029 to 0.24 ± 0.05 at − 40 mV and from 0.52 ± 0.03 to 0.67 ± 0.04 at + 40 mV (Fig. 5D and E). Boltzmann distribution fitting indicated a V1/2 of − 67 ± 1.14 mV in sham rats and 40.5 ± 4.2 mV in STZ rats, with an equivalent gating charge (zd) of − 2.74 ± 0.42 and − 2.34 ± 0.43, respectively. In STZ + light rats, V1/2 was − 45.25 ± 4.1 mV with zd of − 2.74 ± 0.86.

To verify the mitoBKCa channel type, the effects of IbTx, ChTx, and ATP were examined. Channel activity was inhibited by 100 nM IbTx at negative and positive potentials in both STZ and STZ + light rats, but not by 2.5 mM ATP or 500 nM ChTx. These results confirm that the channels were ATP-insensitive mitoBKCa channels (conductance of 211 pS, inhibited only by IbTx; Fig. 5F and G).

Fig. 5
figure 5

Induction of AD reduced channel gating and conductance in mitochondrial mitoBKCa channels but was prevented by 40 Hz light therapy. Single channel recordings in 200/50 mM (cis/trans) KCl solution were performed using a bilayer lipid membrane setup. Single channel current-voltage relationships for mitoBKCa channels and percent of changes in the channel conductance and open probability (Po) as a function of voltage for mitoBKCa channels were evaluated in sham, STZ, and STZ + light animals (A-E). The closed state is marked by the black arrows. Each point represents the average Po as a function of voltages from five different experiments. The effects of IbTx, ChTx, and ATP on mitoBKCa channel activity were determined in STZ (F) and STZ + light animals (G). One-way ANOVA analyses followed by a Bonferroni’s post hoc test were performed to compare the mean between groups. Data are expressed as mean ± SEM (n = 5 (5 A-E) and 4 (5 F-G) rats per group). *P < 0.0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to sham, and #P < 0.05, ##P < 0.01, ####P < 0.0001 compared to STZ.

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Overall, 40 Hz flickering light therapy effectively improved mitoBKCa channel activities in the STZ-induced AD rat model.

Flickering 40 Hz white light counteracted the STZ-Induced disruption in mitochondrial metabolite levels in rat brains

MS-MS was used to evaluate potential changes in the levels of mitochondrial metabolites (amino acids) as a readout of the modified function of mitoBKCa and mitochondrial function. Ten amino acids were significantly reduced in STZ compared to sham brains [tyrosine (P = 0.0006), tryptophan (P = 0.0346), alanine (P = 0.0338), leucine (P = 0.0001), methionine (P = 0.0023), proline (P = 0.0172), lysine (P = 0.0076), serine (P = 0.0237), aspartic acid (P = 0.0499) and glutamic acid (P = 0.0001)]. Interestingly, these reductions were absent in STZ + light brains, and amino acid levels were similar to those in sham controls (Fig. 6A C).

Fig. 6
figure 6

Flickering 40 Hz white light counteracted the STZ-induced disruption in mitochondrial amino acid levels in rat brains. Differential levels of mitochondrial amino acids measured using MS-MS were compared in sham, sham + light, STZ, and STZ + light rats (A-B). Ten significant metabolites were downregulated in STZ rats vs. sham controls (C). A diagram of metabolite-protein joint pathways was generated (D), from which seven common pathways were extracted (E). The top 10 associated pathways affected by alanine, aspartate, and glutamine metabolism are illustrated (F). Data are expressed as mean ± SEM (n = 7 ± 1).

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Metabolites dysregulated in rats with STZ-Induced AD were mostly enriched in pathways that regulate the Krebs cycle (citrate Cycle/TCA), carbohydrate metabolism, and amino acid metabolism in mitochondria

Integrating the results obtained from KEGG and MetaCyc (please see Materials and Methods section) ultimately revealed a list of 154 proteins (Supplementary Table 1). By considering a P-value < 0.05, 10 specific pathways were retrieved after enrichment analysis (Supplementary Table 2), and 8 distinct pathways were identified following pathway analysis (Supplementary Table 3). Moreover, metabolite-protein joint pathway analyses were performed within two contexts: (i) metabolic pathways (integrated), revealing 19 distinct signaling pathways (Supplementary Table 4), and (ii) all pathways (integrated), rendering 39 key pathways (Supplementary Table 5) (Fig. 6D).

A total of seven common key pathways were identified by integrating the four lists (Fig. 6E): alanine, aspartate, and glutamate metabolism (rno00250); arginine biosynthesis (rno00220); histidine metabolism (rno00340); cysteine and methionine metabolism (rno00270); arginine and proline metabolism (rno00330); phenylalanine, tyrosine and tryptophan biosynthesis (rno00400); nitrogen metabolism (rno00910) (Fig. 6E). Of these, the most significant signaling pathway (based on FDR < 0.05) appeared to be alanine, aspartate, and glutamate metabolism (rno00250), which was involved in 10 key pathways (Fig. 6F). Alanine, aspartate, and glutamate metabolism and 10 associated pathways were tightly linked to STZ-induction of experimental AD and beneficial therapeutic effects of flickering 40 Hz white light in our rat model.

Discussion

In this study, we demonstrate that flickering 40 Hz white light has beneficial effects on the brains of rats with STZ-induced AD, suggesting this non-pharmacological, non-invasive approach may hold promise for treating or preventing AD.

STZ injection in rats caused behavioral disorders, mitochondrial dysfunction, plasticity impairment, neuronal death, neurotransmitter changes, and Aβ plaque accumulation, all reminiscent of AD in humans. Exposure to flickering 40 Hz white light improved cognition, dopamine and serotonin levels, and long- and short-term memory in STZ rats. Additionally, flickering 40 Hz white light preserved mitochondrial function, channel current changes, and the opening potential of mitoBKCa channels in rat neurons.

Consistent with previous research on STZ-induced AD models62,63, we observed cognitive memory deficits evidenced by MWM, NOR, and PA test results. Treatment with flickering 40 Hz white light nearly eliminated these STZ-induced impairments. Photomodulation has been suggested to positively regulate cognitive memory in various animal models64,65, including those with experimentally induced AD30,66. Recent studies show that flickering 40 Hz white light ameliorates memory impairments in AD models by inhibiting mitochondrial dysfunction, oxidative stress, and apoptosis67,68. These results support the anti-amnesic potential of flickering 40 Hz white light in an STZ-induced sporadic AD model.

Serotonin and dopamine are crucial neurotransmitters in the brain, which are downregulated in patients with AD and animal models of this neurodegenerative disease69,70,71. Serotonin is the earliest neurotransmitter downregulated, affecting the release of other neurotransmitters, including dopamine72. Light therapy has been shown to decrease serotonin transporter binding, improve serotonin levels, and increase dopamine in humans and rats73,74,75. Our results indicated a modest, albeit insignificant, increase in dopamine in treated groups. Dopamine levels are reduced in the dark and elevated during the light cycle of the day, opposite to melatonin levels16,76,77. Serotonin levels peak in the evening. The increase in serotonin levels in light-treated versus STZ animals may be due to elevated energy supply for synaptic activities, increased intracellular calcium levels, and light-induced neuronal activity78,79.

Decreased neuronal plasticity is a hallmark of neurodegenerative diseases like AD80. Neuronal plasticity is related to synaptic efficacy achieved through long-term potentiation (LTP). LTP is a long-term modification in synaptic strength induced by electrical stimulation, widely used in AD research41,81. To investigate the long-term effects of early 7-day treatment with flickering 40 Hz light and STZ injection into the hippocampus on neuronal activity, we assessed basal synaptic transmission and plasticity in perforant hippocampi 7 days post-STZ injection. Reduced LTP and PPF in the hippocampi of STZ rats may be attributed to compromised NMDA receptor activity and increased Ca2+ influx caused by STZ toxicity42. Similar studies show that short-term plasticity depends on residual Ca2+ in pre-synaptic terminals, with STZ causing calcium imbalance in pre- and post-synaptic regions82. The direct effect of flickering 40 Hz light on the synaptic plasticity of hippocampal CA1 has been demonstrated in mice83. Flickering 40 Hz light may counteract STZ effects on NMDA receptors and modulate Ca2+ levels in cells, preventing suppression of LTP and synaptic plasticity84.

Our histological results indicate overproduction and formation of Aβ plaques in the hippocampus of STZ rats. Accumulating evidence shows that Aβ localizes in mitochondria and interacts with mitochondrial proteins85. Studies indicate that Aβ oligomers can inhibit mitoBKCa channel activity, contributing to mitochondrial dysfunction23,86. Decreased channel open probability and conductance have been reported in STZ-induced early type 2 diabetes models40. In type 2 diabetic mice, insulin resistance in the periphery and brain is associated with energy impairment87,88,89. O’Malley et al. showed that insulin activates mitoBKCa channels in cultured hippocampal cells and modulates spontaneous [Ca2+] oscillations90. Insulin resistance combined with Aβ plaque accumulation in our STZ-induced AD rats likely leads to decreased mitoBKCa channel activity, mitochondrial membrane potential, and mitochondrial dysfunction40. Normally, mitoBKCa channels preserve mitochondrial function and play a neuroprotective role by inhibiting mitochondrial [Ca2+] overload and reducing ROS production36,91. Our finding that intact animal IbTx, but not ATP and ChTx, inhibited channel gating behavior confirms that the channel affected by STZ corresponds to the ATP-insensitive mitoBKCa channel, in line with previous reports37,92. Importantly, flickering 40 Hz white light therapy improved channel activities in our rat model of STZ-induced AD.

Elevation of cytoplasmic calcium can be explained by light-induced opening of calcium ion channels, such as members of the transient receptor potential (TRP) superfamily93. Considering nanostructured water exists in all ion channels and is sensitive to heat and light, mitoBKCa channel phenotype might also be affected by light exposure. Complex IV as an ion channel in the electron transport chain (ETC) might be similarly affected by light treatment94,95. Emitted photons during photomodulation are absorbed by complex IV, facilitating electron availability for oxygen reduction, increasing MMP, ATP levels, and ETC activity96,97. Studies suggest a direct structure-function relationship between the ETC and mitoBKCa channel, proposing that a redox signal from the respiratory chain is transmitted to the mitoBKCa channel through cytochrome c oxidase (complex IV)98.

Studies in mice showed that 590 nm LED light significantly increased NO synthesis in the brain through stimulation of mitochondrial complex IV activity under hypoxic conditions, and that complex IV enzyme activity was inhibited by NO through a feedback mechanism99. Photons emitted by 40 Hz light and absorbed by complex IV can dissociate this inhibitory NO in the presence of sufficient oxygen9. Dissolution of NO results in increased MMP, higher oxygen consumption, glucose metabolism, and more ATP generation. This triggers minimal ROS followed by activation of ROS-induced signaling pathways, which have cytoprotective effects in cells100. Flickering 40 Hz white light might prevent mitochondrial [Ca2+] overload by enhancing ETC and mitoBKCa channel activities91. Optimal mitochondrial [Ca2+] levels prevent ROS overproduction, high MMP, and mitochondrial dysfunction101. This promotes cell viability in the hippocampus and cortex, improving cognition28,102. Furthermore, opening mitoBKCa channels following light treatment keeps calcium ions in the cytoplasm, possibly triggering glutamate release from the presynaptic neuron and improving LTP103,104. Further research is needed to provide more evidence for these mechanisms before definitive conclusions can be drawn.

Gamma oscillations (40 Hz frequency) generated as a result of the interaction between pyramidal cells (1–4 Hz frequency) and interneurons (40 Hz frequency) in the hippocampus play a key role in the regulation of cognition and memory105,106. It is well established that 40 Hz flickering light improves memory in AD patients by generating gamma-oscillations107. Gamma oscillations require mitochondria-generated energy to keep the function of neurons in this region at the peak level ensuring the highest memory performance105. The induction of gamma-oscillations through the 40 Hz flickering light in our study coupled with improved mitochondrial ETC complex activities and mitoBKCa channels might explain the improved memory performance observed in the light treatment group.

Our metabolomics analysis revealed significant decreases of ten mitochondrial amino acids in STZ-induced AD rats, classified into three categories: aromatic (tyrosine, tryptophan), aliphatic (alanine, leucine, methionine, proline), and others (lysine, serine, aspartic acid, glutamic acid). Bioinformatics analyses identified seven common pathways with several affected enzymes/proteins. ‘Alanine, aspartate, and glutamate metabolism’ was the top hit with the lowest FDR and P values. A previous proteomic-metabolomic study in high-sugar-fat diet Wistar rats, an animal model of metabolic syndrome, demonstrated enrichment of three pathways also identified by metabolomics analysis of serum samples from diabetic patients: alanine, aspartate, and glutamate metabolism, glycine, serine, and threonine metabolism, and valine, leucine, and isoleucine biosynthesis108. Transcriptomic and metabolomic joint analysis after an immune challenge with LPS in zebrafish revealed enrichment of ‘alanine, aspartate, and glutamate metabolism’ and aminoacyl-tRNA biosynthesis109. Both studies reported the Krebs cycle as one of the most affected cycles in mitochondria108,109. Our results indicated that TCA and glycolysis, amino acid metabolism, and glutathione metabolism were the top three pathways impacted by ‘alanine, aspartate, and glutamate metabolism.’ The Krebs cycle and glycolysis are key components inside and outside mitochondria for ATP production in neurons and other cells110,111,112. The Krebs cycle is the final hub for lipid, carbohydrate, and amino acid metabolism; disturbance of this cycle is associated with age-related and metabolic diseases108,111,113. During aspartate conversion to asparagine, ammonia is transferred from glutamine to asparagine, converting it to glutamate. Glutamate is used as a substrate to produce α-ketoglutarate and succinate, critical intermediates of the Krebs cycle. Glutamate production and consumption peak in mammals during an immune response114,115,116. Based on our observations, flickering light might reduce Aβ plaque formation by triggering an immune response through microglia activation in the brains of STZ rats, consistent with previous findings in mouse AD models117. Additionally, therapeutic light exposure might induce glutamate metabolism and the Krebs cycle to improve energy production and normalize mitochondrial function114,115,116,117.

Several amino acids, including glutamate, glycine, and cysteine, negatively impact glutathione production, the main ROS scavenger in mitochondria118. Therefore, defects in the metabolism of these amino acids, as indicated in our study, interfere with the ROS-scavenging function of glutathione. This contributes to ROS buildup, likely impeding ETC complex activities and TCA cycle function119. Replenishing glutathione by re-activating glutamatergic neurons and glutamate supply might explain the reducing effect of 40 Hz flickering white light on ROS production19. Furthermore, light therapy might facilitate the transmembrane transport of amino acids and metabolites required for mitochondrial membrane integrity, conductance, and function19.

The majority of ROS is produced by complex I in the mitochondrial ETC120. Mitochondrial membrane integrity helps keep ETC complexes close, increasing respiration efficiency and reducing ROS production121. One explanation for elevated ROS in our rat AD model could be decreased mitochondrial membrane integrity, evidenced by mitochondrial channel conductance and complex I deficiency122. Another possibility could be activation of reverse electron transport (RET) due to disruption in metabolite levels, leading to higher ROS production while keeping ETC activity and mitochondrial function at minimal levels123. Studies have shown that high mitochondrial membrane potential (MMP), accumulation of metabolic substrates, and reduced ubiquinone pools trigger RET123. Our observed high MMP and increased ROS levels associated with aberrant metabolic substrate levels align with this proposed mechanism.

Our findings suggest that 40 Hz flickering light therapy mediates its therapeutic effects primarily through the modulation of mitochondrial function and amino acid metabolism, both of which are critical for synaptic health, neurotransmitter availability, and energy production. In AD, mitochondrial dysfunction is characterized by reduced mitochondrial complex activity, particularly complexes I and IV, and increased ROS production, leading to synaptic failure and neuronal damage124,125. Flickering light therapy restores mitochondrial integrity by enhancing the activity of mitoBKCa channels, which play a key role in maintaining mitochondrial function by regulating calcium influx and ROS production. By preventing calcium overload and reducing mitochondrial ROS levels, flickering light therapy supports ATP production and maintains the efficiency of the ETC. Furthermore, this therapy normalizes the levels of key mitochondrial amino acids—such as glutamate, tryptophan, and tyrosine—which are essential for the Krebs cycle, glutamate-aspartate metabolism, and neurotransmitter synthesis114,115,116,117. These metabolic pathways are closely linked to synaptic plasticity, as they influence the availability of glutamate and serotonin, neurotransmitters critical for synaptic transmission and plasticity.

Additionally, flickering light therapy enhances LTP, a key marker of synaptic plasticity, by improving the availability of neurotransmitters and maintaining the energy demands required for sustained synaptic activity. The improvements in LTP and PPF observed in this study suggest that 40 Hz light therapy can effectively counteract the synaptic plasticity deficits induced by AD.

In conclusion, exposure to 40 Hz flickering white light has great therapeutic potential for treating neurodegenerative disorders such as AD. Our observations strongly support this, showing that daily 15-minute treatments with flickering light for seven consecutive days almost completely counteract the deleterious effects of STZ on cognitive behavior, likely by improving synaptic plasticity and preserving mitochondrial function, in our rat model of STZ-induced AD. While the study provides novel insights into the therapeutic potential of 40 Hz flickering light, there are several limitations. First, we have demonstrated significant improvements in synaptic plasticity and mitochondrial function, but the exact molecular pathways linking mitoBKCa channel activation to improved cognitive outcomes require further elucidation. Future studies could use genetic or pharmacological modulation of these channels to establish a more direct causal relationship. Another limitation of the study was the low exposure time to the 40 Hz flickering light in a limited time frame and the short-term evaluation of the light treatment on memory. Further animal studies are required to examine the longevity of light therapy effects and to establish if long-term changes in plasma membrane BKCa channels occur in parallel with mitoBKCa channels. We also recognize the importance of expanding our investigation into the sex-specific effects of light therapy, as hormonal differences may influence mitochondrial function and synaptic plasticity in a sex-dependent manner. Lastly, while our results in rats are promising, the translatability of 40 Hz flickering light therapy to human AD patients remains to be established. Future clinical studies are necessary to determine whether the beneficial effects observed in animal models can be replicated in human populations.